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Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.

ABSTRACT: Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets.

SUBMITTER: Stanley SA

PROVIDER: S-EPMC3710825 | biostudies-other | 2013 Jul

REPOSITORIES: biostudies-other

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Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.

Stanley Sarah A SA Kawate Tomohiko T Iwase Noriaki N Shimizu Motohisa M Clatworthy Anne E AE Kazyanskaya Edward E Sacchettini James C JC Ioerger Thomas R TR Siddiqi Noman A NA Minami Shoko S Aquadro John A JA Grant Sarah Schmidt SS Rubin Eric J EJ Hung Deborah T DT

Proceedings of the National Academy of Sciences of the United States of America 20130624 28

Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynt ...[more]

PMID: 23798446

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Project description:UnlabelledMycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates.ImportanceMycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

Project description:Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

Dataset Information

Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.

Publications

Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.

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